Http://openwetware.org/wiki/IGEM:IMPERIAL/2006 Engineering a Molecular Predation Oscillator Thanks...

http://openwetware.org/wiki/IGEM:IMPERIAL/2006

Engineering a Engineering a Molecular Predation OscillatorMolecular Predation Oscillator

Thanks to staff from Imperial College for the support over the summer.

Funding

• European Commission• Imperial College Deputy

Rectors Fund• Faculty of Engineering• Faculty of Natural

Sciences

Acknowledgements

Students

• Christin Sander • Deepti Aswani • Farah Vohra • Jiongjun Bai • John Sy • John Chattaway • Jonathan Wells • Tom Hinson

Advisors

• Prof. Richard Kitney • Prof. Paul Freemont • Dr. David Mann • Kirsten Jensen • Vincent Rouilly • Chueh-Loo Poh • Matthieu Bultelle

Team Members

Project Summary Our Favourite PartsAchievements

Our Full System Set-Up

well mixed cultureIn chemostat

[AHL]

Cell population

time

time

Prey molecule generator

Predator molecule generator

Output signal

Wash-out

reservoir

In-flow

Design

• Based on Lotka-Volterra predation dynamics• Use of quorum sensing/quenching BioBricks available from the Registry• Population wide oscillations of AHL in a chemostat• Design broken down into two cell system to introduce higher flexibility

Modelling

• Derivation of the complete dynamical model • Full theoretical analysis and detailed simulations• Existence of oscillations with controllable frequency, amplitude and profile

Implementation

• Standard assembly using BioBricks• Successful building of oscillator components• Contributions to the Registry by adding tested, functional and intermediate parts• Quality control procedure

Testing/Validation

• Definition of testing protocols to satisfy component specifications• Analysis of experimental data• Characterization of the different test constructs for extracting parameters

Specifications

• Stable oscillations for more than 10 periods• High Signal to Noise Ratio• Controllable frequency and amplitude• Modular design for easy connectivity• Full documentation for quality control

Parameters:

a, b, c : Population-dependent a0, b0, c0: Constants d1, d2, e : Wash-out related

• We have used the traditional engineering approach to build a stable and flexible molecular oscillator

• Our original design relies on population dynamics and was inspired by the Lotka-Volterra predation model

• Every step of the development cycle (Specifications, Design, Modelling, Implementation, Testing/Validation) has been fully documented on our OWW site

• Derivation of the complete dynamical model, describing the main biochemical reactions driving our oscillator.

• Full theoretical analysis and detailed computer simulations, validating our design with regard to our specifications.

• Successful building and characterization of functional parts, providing the building blocks for the final oscillator.

PoPs Blocker

• Use of Cre-Recombinase• PoPs control mechanism defining an irreversible switch

Predator Sensing

• Characterization of a new AHL sensing part

Prey Molecule Generator

• Production of AHL via positive feedback loop

Oscillator

On/Off

Frequency tuningAmplitude tuning

Output signal

Oscillator

On/Off

Frequency tuningAmplitude tuning

Output signal

Oscillator

On/Off

Frequency tuningAmplitude tuning

Output signal

Test part

Predictive model transfer function

Experimental data

Fitting model to data

Parameter extractions

[AHL]

[GF

P]

Prey Generator

Prey Sensing

PoPs

AHL

a

a0

Predator Generator

Predator Sensing

PoPs

AHL

c

c0

Predator Killing

AHL

time

b

b0

Our Full System Set-Up

The Prey Generator

LuxI LuxR

RequiredDynamic

UsefulBioBricks

FinalConstruct LuxILuxR

tetR pLux

E.Coli

LuxILuxR

LuxR

Self promoted expression of A

AHLAHL

tetR pLux

The Predator Generator

LuxR aiiANatural

degradation

Degradation of A by B

Expression of B promoted by A/B interaction

Degradation of B

AHLAHL

Lactonase

Killing

LuxR

LuxR

SensingLuxR aiiA

E.ColiAHL

AHL

AHL

pLux

pLux

AHL

well mixed cultureIn chemostat

[AHL]

Cell population

time

time

LuxILuxRtetR pLux

LuxILuxRtetRtetR pLuxpLux

pLuxLuxR aiiA

pLuxpLuxLuxR aiiA

Prey molecule generator

Predator molecule generator

Output signal

Wash-out

reservoir

In-flow

Change in population ratio

J37019

J37032

J37033

J37034

Sensing PreyT9002

Final PreyJ37015

Built Sequenced DocumentedTested Characterized

Built BuiltSequenced Sequenced

Intermediate Parts

Cre/Lox J37027

Sensing PredatorJ37016

J37023

Functional Parts

AHL

LuxRdt

d[LuxR]

dt

d[LuxR]

Gene Expression Enzymatic Reaction

AHLe AHLe

LuxRd1 LuxRd1

Washout

dt

d[AHL]

dt

d[AHL] AHLa

AHLa

0 AHLa

AHLa

0

AHLb

AHLaiiAb

0

AHLb

AHLaiiAb

0

LuxRAHLc

LuxRAHLc

0 LuxRAHLc

LuxRAHLc

0

Self promoted expression of

AHL

Degradation of AHL

Degradation of AHL by aiiA

Production of LuxR

Degradationof LuxR

aiiAdt

d[aiiA]

dt

d[aiiA] LuxRAHLc

LuxRAHLc

0 LuxRAHLc

LuxRAHLc

0 aiiAd2 aiiAd2

Production of aiiA

Degradation of aiiA

Oscillations with limit cycles No oscillations

Prey

Predator

Prey

Time

Predator

Prey

Prey

Time